Nanotechnology is rapidly resulting in the production of nanomaterials (NMs) that will be used in everything from toothpaste to pesticides, yet the research community lacks adequate techniques to measure the size and concentration of nanomaterials in even the simplest of environmental and biological samples at levels which may be relevant to human exposures. The National Nanotechnology Initiative, including NIEHS, and other organizations ranks as a top priority the need to develop methods to quantify nanomaterials in matrices including drinking water, commercial products, blood and other biological matrices. The purpose of our proposed work is two-fold: first, we aim to develop state-of-the- art exposure assessment tools for nanomaterials for the international scientific, medical and regulatory community;second, we will identify and quantify the metrology gap between what exposure levels may be potentially harmful based on published toxicological data and the method detection limits that can be achieved for non-labeled commercial nanomaterials with current technology. Inorganic and carbonaceous nanomaterials are being synthesized in a wide range of sizes, shapes and with various surface coatings or functionalities. Consequently, people may soon be exposed to thousands of different types of nanomaterials in their workplace or during other daily activities. Risk assessments from these exposures are hampered by the lack of adequate detection capabilities. While electron microscopy and other techniques can image NMs in samples, they fall short of being able to quantify the size, number concentration and mass concentration of NMs which are thought to be crucial for understanding to properly assess NM exposures and effects. We hypothesize that two basic instrumentation platforms (ion coupled plasma mass spectroscopy and liquid chromatography mass spectroscopy) can be developed in conjunction with sample pretreatment methods, involving extraction, separation and or concentration of NMs from environmental and biological samples, to quantify the size, number concentration and mass concentration of the currently most widely used inorganic NMs (Ag, TiO2, Au) and carbonaceous NMs (fullerenes and functionalized fullerenes). To support this hypothesis we will exploit techniques initially developed to quantify natural aquatic colloids (i.e., NMs) and organic pollutants. Specifically, real time single particle ICP mass spectroscopy (RTSP-MS) will be used to differentiate metal or metal oxide NMs from dissolved ionic forms of the base NM material. Carbonaceous NMs will be handled in a similar fashion as organic chemicals, by pre-treating and analyzing (LC/MS) them based upon solubility and hydrophobicity characteristics. By working with a range of widely employed NMs the methods will be immediately and widely applicable. Standard operating procedures for NM analytical techniques and extraction protocols will be developed. The investigators have been working with NMs for many years, and are familiar with purchasing, characterizing, solubilizing and handling NMs. Using robust statistical approaches, the procedures (detection limits, precision, accuracy, reproducibility, recovery rate, etc) will be validated. Once validated, the pretreatment methods and analytical techniques will be used to quantify engineered nanomaterials in drinking water, food, consumer products and biological fluids (including whole blood, blood plasma, blood serum, urine and human milk). Our team has extensive experience in the analysis of manmade pollutants in these matrices. Due to a presumably low present ambient exposure to engineered NMs, except perhaps for TiO2, we do not expect to detect these types of materials in our archived samples of biological fluids or drinking waters. After testing this hypothesis, we will fortify aliquots of pools of blood, human milk and urine, respectively, with known and increasing concentrations of diverse engineered NMs, until detection becomes possible. In addition to validating the procedures, this work will establish the present body burden in humans of NMs (or lack thereof) and will help to define what levels of these materials are required in order to achieve detection of engineered NMs with state of the art techniques. All protocols developed will be published in peer- reviewed journals and made freely available on the Internet as step-by-step procedures to enable other laboratories and researchers in the U.S. and abroad to utilize these new human exposure assessment tools. In our data analysis and interpretation, we will compare achievable method detection limits with toxic threshold information to evaluate the prospects of using these novel tools for environmental exposure assessment and for protecting human health.